The interaction between the human microbiome and the host is of increasingly appreciated importance in human biology. The establishment and maintenance of gut microbes after birth assumes importance for many disorders in the neonatal period and later in life, including necrotizing enterocolitis, inflammatory bowel disease, and obesity. As such, the human neonate provides a unique opportunity to study the human intestinal microbiome. Previous attempts to study the developing newborn microbiome have included bacterial culture and 16S rRNA gene sequencing of nucleic acid in the stools of normal infants, but these approaches have limitations. The host and microbial transcriptome, and the aggregate metatranscriptome, can provide a more vivid and complete portrait of the precise metabolic activity of both host epithelial and inflammatory cells, and of the enteric microbial biomass. I propose to use RNA sequencing to characterize the intestinal microbial and host transcriptome in human neonates. We have recently published a new method to isolate and amplifiable host mRNA from human stool. We are also able to isolate and amplify bacterial transcripts. It is now appropriate to determine if RNA analysis in human stool, and most particularly RNA analysis by mass sequencing, illuminates host physiology and pathophysiology, and profiles the enteric microbiome. My first aim will be to develop a computational pipeline to process the mass-sequenced RNA in newborn stool. This will involve a stepwise process of alignment with both human and sequenced microbial genomes, as well as advanced computational methods to ascertain the identity of remaining sequence. My second aim will be to compare the resultant metatranscriptome with metagenomic sequence in the same specimen to verify the hypothesis that the transcript profile is sufficiently different than a genomic census. I will accomplish these goals with a combination of graduate course work in computer sciences and sequential study of a unique neonatal specimen set. PUBLIC HEALTH RELEVANCE: Development of the bacterial population in the intestinal tract is of major importance to human health and disease, both in the newborn period and in later life. Previous efforts have characterized this population by enumerating the bacterial species present and the genes they contain. We believe that a thorough understanding of which genes are expressed and at what times they are expressed, in bacteria and in the host will provide instructive new insights into how the bacterial population of the human gut becomes established, and how the gut matures.